PROJECT SUMMARY Human parvovirus B19 (B19V) is a pathogenic human parvovirus. Productive B19V infection is highly restricted to human erythroid progenitor cells in the bone marrow or fetal liver and induces cell cycle arrest and cell death of the infected cells, which results in a series of hematological disorders. In patients with a high demand for erythrocyte production due to high levels of erythrocyte destruction (e.g., sickle cell disease patients), acute B19V infection can cause transient aplastic crisis. In immunocompromised patients, persistent B19V infection can manifest as pure red-cell aplasia. In the fetus, B19V infection can cause hydrops fetalis, a severe form of anemia. Currently, there are neither vaccines nor anti-virals developed to prevent or treat B19V infection- caused hematological disorders. B19V has a single-stranded DNA genome with two identical inverted terminal repeats (ITRs) at the ends. We have identified the minimal viral DNA replication origin (Ori) of 67 nucleotides at the ITRs, which contains binding domains of the viral large nonstructural protein NS1 and an NS1 nicking site. B19V DNA replication proceeds according to the rolling-hairpin model of parvovirus DNA replication. NS1 nicks the Ori at the nicking site by its N-terminal nickase domain, which is a specific enzymatic reaction during the initiation of viral DNA replication. Our central hypothesis is that inhibition of NS1 nicking of the Ori prevents the initiation of viral DNA replication, and therefore, inhibits virus replication. We have established a fluorophore- based in vitro nicking assay using a single-stranded DNA probe spanning the nicking site and the NS1 nickase. In the proposed research here, we aim, firstly, to establish the fluorophore-based nicking assay in a high throughput format and to screen KU libraries of ~10,000 small molecule compounds for identification of leads that inhibit the NS1 nicking activity in vitro. We will then examine the leads for inhibition of viral DNA replication in B19V-permissive UT7/Epo-S1 cells transfected with a B19V replicative form genome and for antiviral activity in B19V infection of ex vivo expanded human erythroid progenitor cells. Next, we aim to determine the structure of the NS1 nickase, which will be used for structure-based rational design of antivirals in the future. Our long-term goal is to develop effective anti-B19V drugs that can treat B19V infection-caused hematological disorders.